Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

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• 作者：Dustin R. Masser ; David R. Stanford ; Niran Hadad ; Cory B. Giles ; Jonathan D. Wren…
• 刊名：AGE
• 出版年：2016
• 出版时间：June 2016
• 年：2016
• 卷：38
• 期：3
• 全文大小：1,801 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Cell Biology
  Geriatrics and Gerontology
  Molecular Medicine
  
• 出版者：Springer Netherlands
• ISSN：1574-4647
• 卷排序：38

文摘

Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.KeywordsBisulfite sequencingRatDNA methylation